Von Willebrand Factor (VWF) and Factor VIII (FVIII) are key central proteins in the initiation and regulation of hemostasis. Each has its own hereditary deficiency disease, which cause either VWD or hemophilia A, and they circulate together as a non-covalent complex in plasma. This project explores the requirements for the establishment of a regulated secretory pathway for one or both VWF and FVIII proteins within endothelial cells and megakaryocytes. Aim 1 will determine the structural and functional requirements within VWF for the storage of both VWF and FVIII and contrasts these pathways within endothelial cells and platelets. The Weibel-Palade body has an absolute requirement for synthesis of pro-VWF while the alpha granule is permissive if pro-VWF is synthesized. Specific Aim 2 will determine the in vivo potential for synthesis of FVIII in the presence of VWF as a novel therapeutic approach in the treatment of hemophilia A. Transgenic (F8-/-) animals expressing FVIII only in either endothelium or megakaryocytes will be studied to determine the therapeutic advantage of expressing FVIII with VWF. Specific Aim 3 will examine the potential for platelet-specific or endothelial cell-specific FVIII expression as a means to bypass the inhibitory activity of FVIII antibodies. Transgenic animals expressing FVIII in endothelial cells or platelets will be studied within the context of inhibitory antibodies to determine if these sites of expression can "bypass" plasma inhibitors by locally releasing active FVIII in the context of VWF. Specific Aim 4 will determine if the endothelial cell is the physiologic site for expression of FVIII or at least the site synthesizing and storing the FVIII released by the administration of DDAVP. Since the controversy continues as to the site of normal FVIII synthesis, these studies will explore whether the DDAVP releasable pool of FVIII has an absolute requirement for endothelial synthesis of FVIII or whether it can be created in the absence of specific endothelial cell synthesis. Tissue specific knockouts of FVIII production will be carried out in the hepatocyte or in the endothelial cell to determine if these tissue-specific knockouts caused a marked reduction in plasma FVIII. Therefore, we feel that tiiese aims will definitively explore the function of VWF on the in vivo and in vitro intracellular biology of FVIII and provide potential for new therapeutic approaches to the treatment of hemophilia A both in patients with and those without FVIII inhibitors.